Glycoprotein hormone superagonists

ABSTRACT

The invention is directed toward a human glycoprotein hormone having at least one, two, three, four, or five basic amino acids in the α-subunit at positions selected from the group consisting of positions 11, 13, 14, 16, 17, and 20. The inventions is also directed to a human glycoprotein where at least one of the amino acids at position 58, 63, and 69 of the β-subunit of the human thyroid stimulating hormone are basic amino acids. The invention is further directed to a modified human glycoprotein hormone having increased activity over a wild-type human glycoprotein hormone, where the modified human glycoprotein comprises a basic amino acid substituted at a position corresponding to the same amino acid position in a non-human glycoprotein hormone having an increased activity over the wild-type human glycoprotein hormone. The invention is also directed to a method of constructing superactive nonchimeric analogs of human hormones comprising comparing the amino acid sequence of a more active homolog from another species to the human hormone, and selecting superactive analogs from the substituted human hormones. The invention is also directed to nucleic acids encoding the modified human glycoprotein hormones, vectors containing those nucleic acids, and host cells containing those vectors.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates generally to modified glycoproteinhormones. Specifically, this invention relates to modifications to ahuman glycoprotein which create superagonist activity.

[0003] 2. Background Art

[0004] Thyrotropin (thyroid-stimulating hormone, TSH) and thegonadotropins chorionic gonadotropin, (CG), lutropin (luteinizinghormone, LH), and follitropin (follicle-stimulating hormone, FSH)comprise the family of glycoprotein hormones. Each hormone is aheterodimer of two non-covalently linked subunits: α and β. Within thesame species, the amino acid sequence of the α-subunit is identical inall the hormones, whereas the sequence of the β-subunit is hormonespecific. (Pierce, J. G. and Parsons, T. F. “Glycoprotein hormones:structure and function.” Ann. Rev. Biochem. 50:465-495 (1981)). The factthat the sequences of the subunits are highly conserved from fish tomammals implies that these hormones have evolved from a common ancestralprotein (Fontaine Y-A. and Burzawa-Gerard, E. “Esquisse de l'evolutiondes hormones gonadotopes et thyreotropes des vertebres.” Gen. Comp.Endocrinol. 32:341-347 (1977)). Evolutionary changes of these hormonesresulted in certain cases in modification of biological activity (Licht,P. et al. “Evolution of gonadotropin structure and function.” Rec.Progr. Horm. Res., 33:169-248 (1977) and Combarnous, Y. “Molecular basisof the specificity of binding of glycoprotein hormones to theirreceptors.” Endocrine Rev. 13:670-691 (1992)), although, specificstructural determinants modulating biopotency have not been elucidated.For example, human thyroid stimulating hormone (hTSH) and bovine thyroidstimulating hormone (bTSH) share high homology in the α (70%) and β(89%) subunit sequence, but bTSH is 6-10 fold more potent than hTSH(Yamazaki, K. et al. “Potent thyrotropic activity of human chorionicgonadotropin variants in terms of ¹²⁵I incorporation and de novosynthesized thyroid hormone release in human thyroid follicles.” J.Clin. Endocrinol. Metab. 80:473-479 (1995)).

[0005] Glycoprotein hormones are crucial in certain therapies, such asin the treatment of patients with thyroid carcinoma. (See, for example,Meier, C. A., et al., “Diagnostic use of Recombinant Human Thyrotropinin Patients with Thyroid Carcinoma (Phase I/II Study).” J. Clin.Endocrinol. Metabol. 78:22 (1994)). The potential use of human thyroidstimulating hormone (TSH) in the treatment of this disease has beenabandoned due to the potential transmission of Creutzfeldt-Jakobdisease. An alternative to the use of human TSH is the use of bovineTSH, but this approach is very limited since this hormone causesside-effects such as nausea, vomiting, local induration, urticaria, anda relatively high possibility of anaphylactic shock (Meier, C. A., etal.). The lack of bioconsistency of urinary gonadotropins and thelimited efficacy of recombinant glycoprotein hormones justify theirfurther replacement with more effective recombinant analogs. Therefore,there is a need for human-derived glycoprotein hormones as well asagonists of these hormones.

[0006] For example the administration of an agonist of the thyroidstimulating hormone in a particular clinical situation such as thyroidcarcinoma, will enhance the uptake of radioiodine into the carcinoma totreat the disease. Agonists of the thyroid stimulating hormone willcause a greater amount of the radioiodine to be targeted to thecarcinoma, thereby resulting in a more effective treatment.Alternatively, glycoprotein hormones used to induce ovulation can bereplaced with superagonists. This will lower the required dose of thehormone which currently is a major medical problem in fertilitytreatment. (Ben-Rafael, Z., et al. “Pharmacokinetics offollicle-stimulating hormone: clinical significance.” Fertility andSterility 63:689 (1995)). Where the use of wild-type folliclestimulating hormone has led to hyperstimulation and higher rates ofmultiple pregnancies and abortions, apparently by a high number ofhormone molecules stimulating many follicles, a superagonist offollicle-stimulating hormone can be administered to treat theinfertility. The use of an agonist of this modified hormone can resultin a lower frequency of stimulation of multiple follicles since a lowernumber of hormone molecules can be administered to achieve the desiredresult.

[0007] The present invention provides, for the first time, specificamino acid substitutions in human glycoprotein hormones which results inhuman glycoprotein hormone analogs that show a major increase in both invitro and in vivo bioactivity.

SUMMARY OF THE INVENTION

[0008] In accordance with the purpose(s) of this invention, as embodiedand broadly described herein, this invention, in one aspect, provides ahuman glycoprotein hormone comprising at least three basic amino acidsin the α-subunit at positions selected from the group consisting ofpositions 11, 13, 14, 16, 17 and 20.

[0009] The invention further provides a human glycoprotein hormonecomprising at least one basic amino acid in the α-subunit at positionsselected from the group consisting of positions 11, 13, 14, 16, 17 and20.

[0010] In another aspect, the invention provides a modified humanglycoprotein hormone having increased activity over a wild-type humanglycoprotein, wherein the modified human hormone comprises a basic aminoacid substituted at a position corresponding to the same amino acidposition in a non-human glycoprotein hormone having an increasedactivity over the wild-type human glycoprotein.

[0011] In another aspect, the invention provides a method of treating acondition associated with a glycoprotein hormone activity in a subjectcomprising administering a therapeutic amount of the glycoproteinhormone of the present invention to the patient.

[0012] In another aspect, the invention provides a method ofconstructing superactive nonchimeric analogs of human hormonescomprising comparing the amino acid sequence of a more active homologfrom another species to the human hormone, substituting amino acids inthe human hormone with the corresponding amino acids from the otherspecies, determining the activity of the substituted human hormone, andselecting superactive analogs from the substituted human hormones.

[0013] In yet another aspect, the present invention provides nucleicacids which encode the modified glycoprotein hormones.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows a comparison of the relevant primary sequences of theα-subunit from 27 different species (a). Alignment of the subunitsequences obtained from sequencing of PCR amplified fragment of genomicDNA in chimpanzee, orangutan, gibbon and baboon (underlined), receivedfrom GeneBank, SWISS-PROT and PDB databank were made. The numbering ofthe sequences corresponds to that of human α-subunit sequence. Dashes( - - - ) indicate amino acid residues which are identical to those ofthe human α-subunit. Conserved among different species lysine residuesare bolded. The primate sequences determined in this study areunderlined. The human, chimpanzee and orangutan α-subunit sequences arethe only sequences without basic amino acids in this region, despite therelatively high degree of similarity in diverse vertebrate species. (b)Mutations of human sequence made in this region included introduction ofsingle and multiple Lys residues present in all non-human mammaliansequences. Additionally, alanine mutagenesis of residues 13, 16 and 20was used to study the role of Gln13, Pro16 and Gln20.

[0015]FIG. 2 shows the bioactivities and receptor binding activities ofthe most potent hTSH analogs: (a, b) cAMP stimulation in CHO-JP09 cells.Data represent the mean ±SEM of triplicate determinations from arepresentative experiment repeated three (a) and two (b) times. (c, d)Receptor-binding activities to CHO-JP09 cells. The same mutants testedas in the FIG. 2a and FIG. 2b respectively. Values are the mean ±SEM ofquadruplicate determinations from one experiment, repeated two times.(e) Thymidine uptake stimulation in FRTL-5 cells. Values are the mean±SEM of quadruplicate determinations from one experiment, repeated twotimes. (f) Stimulation of T₄ secretion in mice. Each data pointrepresents the mean ±SEM of values from 4-5 animals of a representativeexperiment repeated two times. (g) cAMP stimulation in CHO-hTSH cells.Data represent the mean ±SEM of 3-4 determinations from a representativeexperiment repeated 3 times.(h) Receptor-binding activities in CHO-JP09cells. Data represent the mean ±SEM of 3-4 determinations from arepresentative experiment repeated 3 times.(i) Stimulation of T₄secretion in mice. Each data point represents the mean ±SEM of valuesfrom 4-5 animals of a representative experiment repeated two times.

[0016]FIG. 3 shows the bioactivities and receptor binding activities ofthe most potent hCG analogs. Progesterone production stimulation (a) andreceptor binding assay (b) in MA-10 cells. Data represent the mean ±SEMof triplicate determinations from a representative experiment repeatedthree times. The relative maximal production levels of progesterone arepresented in the Table II as % obtained with WT-hCG. cAMP stimulation(c) and receptor binding assay (d) in COS-7 cells expressing hLHreceptor. Data represent the mean ±SEM of triplicate determinations froma representative experiment repeated two times.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention may be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Example included therein and to the Figures andtheir previous and following description.

[0018] Before the present compounds, compositions, and methods aredisclosed and described, it is to be understood that this invention isnot limited to specific hormones, specific subjects, i.e. humans as wellas non-human mammals, specific amino acids, specific clinicalconditions, specific analogs, or specific methods, as such may, ofcourse, vary, and the numerous modifications and variations therein willbe apparent to those skilled in the art. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting.

[0019] As used in the specification and in the claims, “a” can mean oneor more, depending upon the context in which it is used. Thus, forexample, reference to “a human glycoprotein hormone” means that at leastone human glycoprotein hormone is utilized.

[0020] In one aspect, the invention provides a human glycoproteinhormone comprising at least three basic amino acids in the α-subunit atpositions selected from the group consisting of positions 11, 13, 14,16, 17 and 20.

[0021] The invention further provides a human glycoprotein hormonecomprising at least one basic amino acid in the α-subunit at positionsselected from the group consisting of positions 11, 13, 14, 16, 17 and20.

[0022] In another aspect, the invention provides a modified humanglycoprotein hormone having increased activity over a wild-type humanglycoprotein, wherein the modified human hormone comprises a basic aminoacid substituted at a position corresponding to the same amino acidposition in a non-human glycoprotein hormone having an increasedactivity over the wild-type human glycoprotein.

[0023] In another aspect, the invention provides a method of treating acondition associated with a glycoprotein hormone activity in a subjectcomprising administering a therapeutic amount of the glycoproteinhormone of the present invention to the patient.

[0024] In another aspect, the invention provides a method of assistingreproduction in a subject comprising administering an assisting amountof the glycoprotein hormone of the present invention.

[0025] In another aspect, the invention provides a method ofconstructing superactive nonchimeric analogs of human hormonescomprising comparing the amino acid sequence of a more active homologfrom another species to the human hormone, substituting amino acids inthe human hormone with the corresponding amino acids from the otherspecies, determining the activity of the substituted human hormone, andselecting superactive analogs from the substituted human hormones.

[0026] By “human” glycoprotein hormone is meant that the number of aminoacid substitutions made in the wild-type sequence does not exceedone-half the number of amino acid differences at corresponding positionsin the corresponding polypeptide hormones between human and anotherspecies. Thus, the modified polypeptide hormone would be considered morelike the wild-type polypeptide hormone of the human than thecorresponding polypeptide hormone from the non-human species from whichthe amino acid substitutions are derived, based on the amino acid codingsequence. For example, if there were a total of 20 amino aciddifferences at corresponding positions in corresponding glycoproteinhormones between a human glycoprotein and a bovine glycoprotein hormone,a “human” glycoprotein hormone would be a modified wild-type humanhormone which contains 10 or fewer amino acid substitutions within itsamino acid sequence which are homologous to the corresponding aminoacids in the bovine amino acid sequence. More specifically, the thyroidstimulating hormone, as set forth in the Examples contained herein,would be considered “human” if 20 or more of the 40 total amino aciddifferences between the α- and β-subunits of the human and the bovinehomologs are homologous to the amino acid at the corresponding positionin the human thyroid stimulating hormone.

[0027] Naturally, because of the risk of an adverse immune response tothe administration of the modified glycoprotein hormone where therecipient of the modified glycoprotein hormone is a human, the modifiedglycoprotein hormone is preferably homologous to the human amino acidsequence to the greatest extent possible without an unacceptable loss inthe superagonist activity. Alternatively, where the subject beingadministered the modified glycoprotein is non-human, the modifiedglycoprotein hormone is preferably homologous to the specific non-humanamino acid sequence to the greatest extent possible without anunacceptable loss in the superagonist activity. Thus, in a preferredembodiment of the present invention, in modifying a wild-typeglycoprotein to construct a modified glycoprotein with a superagonistactivity by substituting specific amino acids, the substituted aminoacids which do not increase agonist activity number 10 or less,especially 9, 8, 7, 6, 5, 4, 3, and 2 or zero.

[0028] Likewise, by “nonchimeric” is meant that the number of aminosubstitutions does not exceed one-half the number of amino aciddifferences at corresponding positions in the corresponding polypeptidehormones between species, such that the modified polypeptide hormonewould be considered more like the wild-type polypeptide hormone of thespecies being modified than the corresponding polypeptide hormone fromthe species from which the amino acid substitutions are derived, basedon the amino acid coding sequence.

[0029] In yet another aspect, the present invention provides nucleicacids which encode the modified glycoprotein hormones.

[0030] Glycoprotein hormones comprise a family of hormones which arestructurally related heterodimers consisting of a species-commonα-subunit and a distinct β-subunit that confers the biologicalspecificity for each hormone. For a general review of glycoproteinhormones, see Pierce, J. G. et al., “Glycoprotein hormones: structureand function.” Ann. Rev. Biochem. 50:465-495 (1981), see alsoCombarnous, Y. “Molecular basis of the specificity of binding ofglycoprotein hormones to their receptors.” Endocrine Rev. 13:670-691(1992) This family of hormones includes chorionic gonadotropin (CG),lutropin (luteinizing hormone, LH), follitropin (follicle-stimulatinghormone, FSH), and thyrotropin (thyroid-stimulating hormone, TSH). Eachof these glycoprotein hormones with at least one basic amino acid in theα-subunit at positions selected from the group consisting of positions11, 13, 14, 16, 17, and 20, is provided by the present invention.

[0031] Basic amino acids comprise the amino acids lysine, arginine, andhistidine, and any other basic amino acid which may be a modification toany of these three amino acids, synthetic basic amino acids not normallyfound in nature, or any other amino acid which is positively charged ata neutral pH.

[0032] The glycoprotein hormones provided for by the present inventionmay be obtained in any number of ways. For example, a DNA moleculeencoding a glycoprotein hormone can be isolated from the organism inwhich it is normally found. For example, a genomic DNA or cDNA librarycan be constructed and screened for the presence of the nucleic acid ofinterest. Methods of constructing and screening such libraries are wellknown in the art and kits for performing the construction and screeningsteps are commercially available (for example, Stratagene CloningSystems, La Jolla, Calif.). Once isolated, the nucleic acid can bedirectly cloned into an appropriate vector, or if necessary, be modifiedto facilitate the subsequent cloning steps. Such modification steps areroutine, an example of which is the addition of oligonucleotide linkerswhich contain restriction sites to the termini of the nucleic acid.General methods are set forth in Sambrook et al., “Molecular Cloning, aLaboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

[0033] Once the nucleic acid sequence of the desired glycoproteinhormone is obtained, basic amino acids can be positioned at anyparticular amino acid positions by techniques well known in the art. Forexample, PCR primers can be designed which span the amino acid positionor positions and which can substitute a basic amino acid for a non-basicamino acid. Then a nucleic acid can be amplified and inserted into thewild-type glycoprotein hormone coding sequence in order to obtain any ofa number of possible combinations of basic amino acids at any positionof the glycoprotein hormone. Alternatively, one skilled in the art canintroduce specific mutations at any point in a particular nucleic acidsequence through techniques for point mutagenesis. General methods areset forth in Smith, M “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462(1985) and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605-610 (1991).

[0034] Another example of a method of obtaining a DNA molecule encodinga specific glycoprotein hormone is to synthesize a recombinant DNAmolecule which encodes the glycoprotein hormone. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein region are readilyobtainable through automated DNA synthesis. A nucleic acid for onestrand of a double-stranded molecule can be synthesized and hybridizedto its complementary strand. One can design these oligonucleotides suchthat the resulting double-stranded molecule has either internalrestriction sites or appropriate 5′ or 3′ overhangs at the termini forcloning into an appropriate vector. Double-stranded molecules coding forrelatively large proteins can readily be synthesized by firstconstructing several different double-stranded molecules that code forparticular regions of the protein, followed by ligating these DNAmolecules together. For example, Cunningham, et al., “Receptor andAntibody Epitopes in Human Growth Hormone Identified by Homolog-ScanningMutagenesis,” Science, 243:1330-1336 (1989), have constructed asynthetic gene encoding the human growth hormone gene by firstconstructing overlapping and complementary synthetic oligonucleotidesand ligating these fragments together. See also, Ferretti, et al., Proc.Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pairsynthetic bovine rhodopsin gene from synthetic oligonucleotides isdisclosed. By constructing a glycoprotein hormone in this manner, oneskilled in the art can readily obtain any particular glycoproteinhormone with basic amino acids at any particular position or positionsof either the α-subunit, the β-subunit, or both. See also, U.S. Pat. No.5,503,995 which describes an enzyme template reaction method of makingsynthetic genes. Techniques such as this are routine in the art and arewell documented. DNA fragments encoding glycoprotein hormones can thenbe expressed in vivo or in vitro as discussed below.

[0035] Once a nucleic acid encoding a particular glycoprotein hormone ofinterest, or a region of that nucleic acid, is constructed, modified, orisolated, that nucleic acid can then be cloned into an appropriatevector, which can direct the in vivo or in vitro synthesis of thatwild-type and/or modified glycoprotein hormone. The vector iscontemplated to have the necessary functional elements that direct andregulate transcription of the inserted gene, or hybrid gene. Thesefunctional elements include, but are not limited to, a promoter, regionsupstream or downstream of the promoter, such as enhancers that mayregulate the transcriptional activity of the promoter, an origin ofreplication, appropriate restriction sites to facilitate cloning ofinserts adjacent to the promoter, antibiotic resistance genes or othermarkers which can serve to select for cells containing the vector or thevector containing the insert, RNA splice junctions, a transcriptiontermination region, or any other region which may serve to facilitatethe expression of the inserted gene or hybrid gene. (See generally,Sambrook et al.).

[0036] There are numerous E. coli (Escherichia coli) expression vectorsknown to one of ordinary skill in the art which are useful for theexpression of the nucleic acid insert. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxy-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

[0037] Additionally, yeast expression can be used. There are severaladvantages to yeast expression systems. First, evidence exists thatproteins produced in a yeast secretion systems exhibit correct disulfidepairing. Second, post-translational glycosylation is efficiently carriedout by yeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, et al.,“∝-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins inSaccharomyces cerevisiae.” Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).The leader region of pre-pro-alpha-factor contains a signal peptide anda pro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene: this enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. Thenucleic acid coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.Efficient post translational glycosolation and expression of recombinantproteins can also be achieved in Baculovirus systems.

[0038] Mammalian cells permit the expression of proteins in anenvironment that favors important post-translational modifications suchas folding and cysteine pairing, addition of complex carbohydratestructures, and secretion of active protein. Vectors useful for theexpression of active proteins in mammalian cells are characterized byinsertion of the protein coding sequence between a strong viral promoterand a polyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation maybe used for othercellular hosts.

[0039] Alternative vectors for the expression of genes in mammaliancells, those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VRIII,hepatitis B virus surface antigen, protease Nexinl, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

[0040] Expression of the gene or hybrid gene can be by either in vivo orin vitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector. An exampleof modified glycoprotein hormones inserted into a prokaryotic expressionvector is given in the Example section contained herein.

[0041] Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of mRNA. In such in vitro transcription systems, thenucleic acid encoding the glycoprotein hormone would be cloned into anexpression vector adjacent to a transcription promoter. For example, theBluescript II cloning and expression vectors contain multiple cloningsites which are flanked by strong prokaryotic transcription promoters.(Stratagene Cloning Systems, La Jolla, Calif.). Kits are available whichcontain all the necessary reagents for in vitro synthesis of an RNA froma DNA template such as the Bluescript vectors. (Stratagene CloningSystems, La Jolla, Calif.). RNA produced in vitro by a system such asthis can then be translated in vitro to produce the desired glycoproteinhormone. (Stratagene Cloning Systems, La Jolla, Calif.).

[0042] Another method of producing a glycoprotein hormone is to link twopeptides or polypeptides together by protein chemistry techniques. Forexample, peptides or polypeptides can be chemically synthesized usingcurrently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to a hybrid glycoprotein hormone can be synthesized bystandard chemical reactions. For example, a peptide or polypeptide canbe synthesized and not cleaved from its synthesis resin whereas theother fragment of a hybrid peptide can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. By peptide condensationreactions, these two fragments can be covalently joined via a peptidebond at their carboxyl and amino termini, respectively, to form a hybridpeptide. (Grant, G. A., “Synthetic Peptides: A User Guide,” W.H. Freemanand Co., N.Y. (1992) and Bodansky, M. and Trost, B., Ed., “Principles ofPeptide Synthesis,” Springer-Verlag Inc., N.Y. (1993)). Alternatively,the peptide or polypeptide can by independently synthesized in vivo asdescribed above. Once isolated, these independent peptides orpolypeptides may be linked to form a glycoprotein hormone via similarpeptide condensation reactions.

[0043] For example, enzymatic ligation of cloned or synthetic peptidesegments can allow relatively short peptide fragments to be joined toproduce larger peptide fragments, polypeptides or whole protein domains(Abrahmsen, L., et al., Biochemistry, 30:4151 (1991)). Alternatively,native chemical ligation of synthetic peptides can be utilized tosynthetically construct large peptides or polypeptides from shorterpeptide fragments. This method consists of a two step chemical reaction(Dawson, et al., “Synthesis of Proteins by Native Chemical Ligation,”Science, 266:776-779 (1994)). The first step is the chemoselectivereaction of an unprotected synthetic peptide-∝-thioester with anotherunprotected peptide segment containing an amino-terminal Cys residue togive a thioester-linked intermediate as the initial covalent product.Without a change in the reaction conditions, this intermediate undergoesspontaneous, rapid intramolecular reaction to form a native peptide bondat the ligation site. Application of this native chemical ligationmethod to the total synthesis of a protein molecule is illustrated bythe preparation of human interleukin 8 (IL-8) (Clark-Lewis, I., et al.,FEBS Lett., 307:97 (1987), Clark-Lewis, I., et al., J.Biol.Chem.,269:16075 (1994), Clark-Lewis, L, et al., Biochemistry, 30:3128 (1991),and Rajarathnam, K, et al., Biochemistry, 29:1689 (1994)).

[0044] Alternatively, unprotected peptide segments can be chemicallylinked where the bond formed between the peptide segments as a result ofthe chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M,et al., Science, 256:221 (1992)). This technique has been used tosynthesize analogs of protein domains as well as large amounts ofrelatively pure proteins with full biological activity (deLisle Milton,R. C., et al., “Techniques in Protein Chemistry IV,” Academic Press, NewYork, pp. 257-267 (1992)).

[0045] The invention also provides fragments of modified glycoproteinhormones which have either superagonist or antagonist activity. Thepolypeptide fragments of the present invention can be recombinantproteins obtained by cloning nucleic acids encoding the polypeptide inan expression system capable of producing the polypeptide fragmentsthereof. For example, one can determine the active domain of aglycoprotein hormone which, together with a β-subunit, can interact witha glycoprotein hormone receptor and cause a biological effect associatedwith the glycoprotein hormone. In one example, amino acids found to notcontribute to either the activity or the binding specificity or affinityof the glycprotein hormone can be deleted without a loss in therespective activity.

[0046] For example, amino or carboxy-terminal amino acids can besequentially removed from either the native or the modified glycoproteinhormone and the respective activity tested in one of many availableassays. In another example, a fragment of a modified glycoprotein cancomprise a modified hormone wherein at least one amino acid has beensubstituted for the naturally occurring amino acid at specific positionsin either the α or the β-subunit, and a portion of either amino terminalor carboxy terminal amino acids, or even an internal region of thehormone, has been replaced with a polypeptide fragment or other moiety,such as biotin, which can facilitate in the purification of the modifiedglycoprotein hormone. For example, a modified glycoprotein can be fusedto a maltose binding protein, through either peptide chemistry ofcloning the respective nucleic acids encoding the two polypeptidefragments into an expression vector such that the expression of thecoding region results in a hybrid polypeptide. The hybrid polypeptidecan be affinity purified by passing it over an amylose affinity column,and the modified glycoprotein can then be separated from the maltosebinding region by cleaving the hybrid polypeptide with the specificprotease factor Xa. (See, for example, New England Biolabs ProductCatalog, 1996, pg. 164:).

[0047] Active fragments of a glycoprotein hormone can also besynthesized directly or obtained by chemical or mechanical disruption oflarger glycoprotein hormone. An active fragment is defined as an aminoacid sequence of at least about 5 consecutive amino acids derived fromthe naturally occurring amino acid sequence, which has the relevantactivity, e.g., binding or regulatory activity.

[0048] The fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the peptide is not significantly altered orimpaired compared to the modified glycoprotein hormone. Thesemodifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, etc. In any case, the peptide must possess a bioactiveproperty, such as binding activity, regulation of binding at the bindingdomain, etc. Functional or active regions of the glycoprotein hormonemay be identified by mutagenesis of a specific region of the hormone,followed by expression and testing of the expressed polypeptide. Suchmethods are readily apparent to a skilled practitioner in the art andcan include site-specific mutagenesis of the nucleic acid encoding thereceptor. (Zoller, M. J. et al.).

[0049] In one embodiment of the present invention, the humanglycoprotein hormone comprises at least one basic amino acid in theα-subunit at the position selected from the group consisting ofpositions 11, 13, 14, 16, 17, and 20. In one embodiment, the humanglycoprotein hormone has a basic amino acid at position 11. In anotherembodiment, the human glycoprotein hormone has a basic amino acid atposition 13. In another embodiment, the human glycoprotein hormone has abasic amino acid at position 14. In another embodiment, the humanglycoprotein hormone has a basic amino acid at position 16. In anotherembodiment, the human glycoprotein hormone has a basic amino acid atposition 17. In another embodiment, the human glycoprotein hormone has abasic amino acid at position 20. In another embodiment of the presentinvention, the basic amino acid at position 11, 13, 14, 16, and 20 islysine. In yet another embodiment of the present invention, the basicamino acid at position 17 is arginine.

[0050] The present invention also provides for a human glycoproteinhormone with basic amino acids in the α-subunit in all combinations ofany two positions selected from the group consisting of positions 11,13, 14, 16, 17, and 20. For example, basic amino acids may be present atpositions 11 and 13, or positions 11, and 14, or positions 11 and 16, orpositions 11 and 17, or positions 11 and 20, or positions 13 and 14, orpositions 13 and 17, or positions 14 and 16, or positions 14 and 17, orpositions 14 and 20, or positions 16 and 17, or positions 17 and 20. Inone embodiment of the present invention, the human glycoprotein hormonehas basic amino acids at position 16 and 13. In another embodiment ofthe present invention, the human glycoprotein hormone has basic aminoacids at positions 20 and 13. In yet another embodiment, the humanglycoprotein hormone has basic amino acids at positions 16 and 20.

[0051] The present invention also provides for a human glycoproteinhormone with basic amino acids in the α-subunit in all combinations ofany three positions selected from the group consisting of positions 11,13, 14, 16, 17, and 20. For example, basic amino acids may be present atpositions 11, 13, and 14, or positions 11, 13, and 16, or positions 11,13, and 17, or positions 11, 13, and 20, or positions 11, 14, and 16, orpositions 11, 14, and 17, or positions 11, 14, and 20, or positions 11,16, and 17, or positions 11, 16, and 20, or positions 11, 17, and 20, orpositions 13, 14, and 16, or positions 13, 14, and 17, or positions 13,14, and 20, or positions 13, 16, and 17, or positions 13, 17, and 20, orpositions 14, 16, and 17, or positions 14, 16, and 20, or positions 14,17, and 20, or positions 16, 17, and 20. In a preferred embodiment ofthe present invention, the human glycoprotein hormone has basic aminoacids at positions 13, 16, and 20. In another embodiment of the presentinvention, the hormone is thyroid stimulating hormone. In anotherembodiment of the present invention, the hormone is follicle-stimulatinghormone. In another embodiment of the present invention, the hormone isluteinizing hormone. In another embodiment of the present invention, thehormone is chorionic gonadotropin. In yet another embodiment of thepresent invention, the basic amino acids at any three positions selectedfrom the group consisting of positions 11, 13, 14, 16, 17, and 20, arelysine.

[0052] The present invention also provides for a human thyroidstimulating hormone with at least three basic amino acids in theα-subunit at positions selected from the group consisting of positions11, 13, 14, 16, 17, and 20, where the thyroid stimulating hormone alsohas a basic amino acid in at least one position selected from the groupconsisting of positions 58, 63, and 69 of the β-subunit. In oneembodiment of the present invention, the thyroid stimulating hormone hasa basic amino acid in at position 58 of the β-subunit. In anotherembodiment of the present invention, the thyroid stimulating hormone hasa basic amino acid in at position 63 of the β-subunit. In anotherembodiment of the present invention, the thyroid stimulating hormone hasa basic amino acid in at position 69 of the β-subunit. In anotherembodiment of the present invention, the thyroid stimulating hormone hasa basic amino acid in each of positions 58, 63, and 69 of the β-subunit.In yet another embodiment of the present invention, the basic amino acidin at least one position selected from the group consisting of positions58, 63, and 69 of the β-subunit is arginine.

[0053] The present invention also provides for a human glycoproteinhormone with basic amino acids in the α-subunit in all combinations ofany four positions selected from the group consisting of positions 11,13, 14, 16, 17, and 20. One skilled in the art will readily determinethe possible combinations available. In one embodiment, the humanglycoprotein hormone has basic amino acids at positions 11, 13, 16, and20. In another embodiment, the human glycoprotein hormone has basicamino acids at positions 11, 13, 17, and 20. In another embodiment, thehuman glycoprotein hormone has basic amino acids at positions 13, 14,17, and 20. In a preferred embodiment, the human glycoprotein hormonehas basic amino acids at positions 13, 14, 16, and 20. In yet anotherembodiment of the present invention, the basic amino acids at any fourpositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20, are lysine.

[0054] The present invention also provides for a human glycoproteinhormone with basic amino acids in the α-subunit in all combinations ofany five positions selected from the group consisting of positions 11,13, 14, 16, 17, and 20. One skilled in the art will readily determinethe possible combinations available. In one embodiment, the humanglycoprotein hormone has basic amino acids at positions 13, 14, 16, 17,and 20. In another embodiment, the human glycoprotein hormone has basicamino acids at positions 11, 13, 14, 16, and 20. In yet anotherembodiment of the present invention, the basic amino acids at any fivepositions selected from the group consisting of positions 11, 13, 14,16, 17, and 20, are selected from the group consisting of lysine andarginine.

[0055] The present invention also provides for a human glycoproteinhormone with basic amino acids in the α-subunit in all six of positions11, 13, 14, 16, 17, and 20.

[0056] In another aspect, the present invention provides a humanglycoprotein hormone with a basic amino acid in the α-subunit in atleast one position selected from the group consisting of positions 11,13, 14, 16, 17, and 20, wherein the hormone is human thyroid stimulatinghormone and there is a basic amino acid in at least one positionselected from the group consisting of positions 58, 63, and 69 of theβ-subunit. In one embodiment of the present invention, the humanglycoprotein hormone has a basic amino acid at position 58 of theβ-subunit of the human thyroid stimulating hormone. In anotherembodiment of the present invention, the human glycoprotein hormone hasa basic amino acid at position 63 of the β-subunit of the human thyroidstimulating hormone. In a preferred embodiment of the present invention,the human glycoprotein hormone has a basic amino acid at position 69 ofthe β-subunit of the human thyroid stimulating hormone. In anotherembodiment of the present invention, the human glycoprotein hormone hasbasic amino acids at position 58, 63, and 69 of the β-subunit of thehuman thyroid stimulating hormone. In yet another embodiment of thepresent invention, the basic amino acid at the position selected fromthe group consisting of positions 58, 63, and 69 is arginine.

[0057] In another aspect, the present invention provides a humanfollicle-stimulating hormone, a human luteinizing hormone, or a humanchorionic gonadotropin glycoprotein hormone, wherein the hormonecomprises a basic amino acid in at least one position selected from thegroup consisting of positions in the β-subunit of any of theglycoprotein hormones, corresponding to positions 58, 63, and 69 of theβ-subunit of the human thyroid stimulating hormone. This approachapplies equally to non-humans as well. For example, the β-subunit aminoacid sequences of two bovine glycoprotein hormones can be compared andsubstitutions made to any of the subunits based on the sequencedifferences.

[0058] One skilled in the art can readily determine which sites of theβ-subunits of the other glycoprotein hormones correspond to sites 58,63, and 69 of the β-subunit of the human thyroid stimulating hormone.For example, see Ward, et al., In: Bellet, D and Bidard, J. M. (eds)“Structure-function relationships of gonadotropins” Serono SymposiumPublications, Raven Press, New York, 65:1-19 (1990), where the aminoacid sequences of 26 various glycoprotein hormone β-subunits are alignedand compared. Therefore, one skilled in the art can readily substitutenon-basic amino acids at these sites of the other glycoprotein hormonesfor basic amino acids.

[0059] Similarly, the present invention provides for any humanglycoprotein, wherein the hormone comprises a basic amino acid in atleast one position selected from the group consisting of positions inthe β-subunit of a glycoprotein hormone corresponding to the samepositions in any of the other human glycoprotein hormones. For example,the amino acid sequence of the β-subunits of the human luteinizinghormone and the human chorionic gonadotropin hormone can be compared andamino acid substitutions made at selected sites in either of theseglycoptotein hormones based on the amino acid differences between thetwo β-subunits. This approach also applies equally to non-humans aswell.

[0060] The present invention also provides a modified human glycoproteinhormone having increased activity over a wild-type human glycoprotein,wherein the modified human hormone comprises a basic amino acidsubstituted at a position corresponding to the same amino acid positionin a non-human glycoprotein hormone having an increased activity overthe wild-type human glycoprotein.

[0061] The non-human glycoprotein hormone having an increased activityover the wild-type human glycoprotein can be any non-human species. Forexample, the non-human species can be bovine. See, for example, Benua,R. S., et al “An 18 year study of the use of beef thyrotropin toincrease I-131 uptake in metastatic thyroid cancer.” J. Nucl. Med.5:796-801 (1964) and Hershman, J. M., et al. Serum thyrotropin (TSH)levels after thyroid ablation compared with TSH levels after exogenousbovine TSH: implications for 1-131 treatment of thyroid carcinoma.” J.Clin. Endocrinol. Metab. 34:814-818 (1972). Alternatively, the non-humanspecies can be equine, porcine, ovine, and the like. In the Examplecontained herein, the sequence of the 10-21 amino acid region of 27species is set forth.

[0062] The present invention also provides a modified glycoproteinhormone having increased activity over a wild-type glycoprotein hormonefrom the same species, wherein the modified glycoprotein hormonecomprises a basic amino acid substituted at a position corresponding tothe same amino acid position in a glycoprotein hormone from anotherspecies having an increased activity over the wild-type glycoproteinhormone. Therefore the glycoprotein being modified to increase itsactivity can be from a non-human species. For example, one can compareporcine glycoprotein hormones to bovine glycoprotein hormones, designporcine glycoprotein hormones with amino acid substitutions at positionswhere the porcine and the bovine sequences are different, constructporcine glycoprotein hormones with the selected changes, and administerthe modified porcine glycoprotein hormone to porcine animals.Alternatively, the glycoprotein hormone being modified can be bovine.

[0063] The present invention also provides a modified glycoproteinhormone having increased activity over the wild-type glycoproteinhormone from the same species, wherein the modified glycoprotein hormonecomprises a basic amino acid substituted at a position corresponding tothe same amino acid position in a different glycoprotein hormone fromthe same species having an increased activity over the wild-typeglycoprotein hormone. For example, the β-subunits of humanthyroid-stimulating hormone and human chorionic gonadotropin can becompared and amino acid substitutions to either of these β-subunits canbe made based on any sequence divergence. Naturally, only those changeswhich generally increase or decrease the activity of the modifiedglycoprotein hormone are contemplated since the hormone receptorspecificity will still need to be retained. An example of such aβ-subunit modification is set forth in the Examples contained herein,where basic amino acids were substituted at positions 58 and 63 of thehuman thyroid stimulating hormone based on sequence comparison betweenthe human thyroid stimulating hormone and the human chorionicgonadotropin hormone.

[0064] Modification refers to the substitution of a non-basic amino acidat any particular position or positions of the wild-type glycoproteinwith a basic amino acid. In a presently preferred embodiment of thepresent invention, these modifications comprise the substitution oflysine for a non-basic amino acid.

[0065] The effect of the modification or modifications to the wild-typeglycoprotein hormone can be ascertained in any number of ways. Forexample, cyclic AMP (cAMP) production in cells transfected with themodified glycoprotein can be measured and compared to the cAMPproduction of similar cells transfected with the wild-type glycoproteinhormone. Alternatively, progesterone production in cells transfectedwith the modified glycoprotein can be measured and compared to theprogesterone production of similar cells transfected with the wild-typeglycoprotein hormone. Alternatively, the activity of a modifiedglycoprotein hormone can be determined from receptor binding assays,from thymidine uptake assays, or from T₄ secretion assays. Specificexamples of such assays for determining the activity of modifiedglycoprotein hormones is set forth in the Example section containedherein. One skilled in the art can readily determine any appropriateassay to employ to determine the activity of either a wild-type or amodified glycoprotein hormone.

[0066] In one embodiment of the present invention, the modifiedglycoprotein hormone has an activity which is increased over theactivity of the wild type glycoprotein hormone by at least 3 fold. Thisincreased activity can be assessed by any of the techniques mentionedabove and described in the Example contained herein, or in any otherappropriate assay as readily determined by one skilled in the art. Theincreased activity does not have to be consistent from assay to assay,or from cell line to cell line, as these of course, will vary. Forexample, and as set forth in the Example contained herein, the relativepotency of the P16K mutation in the α-subunit of the human glycoproteinhormone compared to the activity of the wild type glycoprotein hormonein a cAMP assay was approximately 6.4 fold higher. In the progesteronerelease assay, however, the difference between the same mutant and thewild-type glycoprotein hormone was approximately 3.4 fold in potency and1.6 fold in Vmax. This specific modification demonstrates at least a 3fold increase in activity in at least one assay, and thereforerepresents a glycoprotein hormone with at least a 3 fold increase inactivity.

[0067] To modify additional amino acid positions, glycoprotein hormonesequences from human and non-humans can be aligned using standardcomputer software programs such as DNASIS (Hitachi Software EngineeringCo. Ltd.). The amino acid residues that differ between the human and thenon-human glycoprotein hormone can then be substituted using one of theabove-mentioned techniques, and the resultant glycoprotein hormoneassayed for its activity using one of the above-mentioned assays.

[0068] The subject being treated or administered a modified glycoproteinhormone can be a human or any non-human mammal. For example, themodified glycoprotein hormone superagonists may be used in thesuperovulation of bovine animals by administering these glycoproteinhormones to those bovine animals.

[0069] The methods used in substituting a basic amino acid for anon-basic amino acid at any particular position or positions can also beused to design glycoprotein hormone antagonists. By making specificsubstitutions and monitoring the activity of these modified glycoproteinhormones, one can determine which modifications yield glycoproteinhormones with reduced activity. These glycoprotein hormone agonists canbe used in studies of the hormone receptor such as receptor turnoverrates, receptor affinity for the glycoprotein hormone, or even intherapeutic procedures such as treatment of Grave's disease and infertility control.

[0070] The present invention also provides a method of treating acondition associated with a glycoprotein hormone activity in a subjectcomprising administering a therapeutic amount of the glycoproteinhormone of the present invention to the subject. These conditionsinclude any condition associated with a glycoprotein hormone activity.Examples of these conditions include, but are not limited to, ovulatorydisfunction, luteal phase defect, unexplained infertility, male factorinfertility, time-limited conception.

[0071] In another example, the glycoprotein hormone may be administeredto diagnose and treat a thyroid carcinoma. For example, theadministration of bovine TSH to a human subject can be used to stimulatethe uptake of ¹³¹I in thyroid tissue to treat thyroid carcinoma. (Meier,C. A., et al., “Diagnostic use of Recombinant Human Thyrotropin inPatients with Thyroid Carcinoma (Phase I/II Study).” J. Clin.Endocrinol. Metabol. 78:22 (1994)).

[0072] A skilled practitioner in the art can readily determine theeffective amount of the glycoprotein hormone to administer and willdepend on factors such as weight, size, the severity of the specificcondition, and the type of subject itself. The therapeutically effectiveamount can readily be determined by routine optimization procedures. Thepresent invention provides glycoprotein hormones with increased activityrelative to the wild-type glycoprotein hormone. These modifiedglycoprotein hormones will allow a skilled practitioner to administer alower dose of a modified glycoprotein hormone relative to the wild-typeglycoprotein hormones to achieve a similar therapeutic effect, oralternatively, administer a dose of the modified glycoprotein hormonesimilar to the dose of the wild-type glycoprotein hormone to achieve anincreased therapeutic effect.

[0073] Depending on whether the glycoprotein hormone is administeredorally, parenterally, or otherwise, the administration of theprostaglandin can be in the form of solid, semi-solid, or liquid dosageforms, such as, for example, tablets, pills, capsules, powders, liquids,creams, and suspensions, or the like, preferably in unit dosage formsuitable for delivery of a precise dosage. The glycoprotein hormone mayinclude an effective amount of the selected glycoprotein hormone incombination with a pharmaceutically acceptable carrier and, in addition,may include other medicinal agents, pharmaceutical agents, carriers,adjuvants, diluents, etc. By “pharmaceutically acceptable” is meant amaterial that is not biologically or otherwise undesirable, i.e., thematerial may be administered to an individual along with the selectedglycoprotein hormone without causing unacceptable biological effects orinteracting in an unacceptable manner with the glycoprotein hormone.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, latest edition (Mack Publishing Co., Easton,Pa.).

[0074] In another aspect, the present invention provides a method ofassisting reproduction in a subject comprising administering anassisting amount of the glycoprotein hormone of the present invention.For example, in a subject with isolated gonadotropin deficiency (IGD),administration of modified follicle stimulating hormone (follitropin)and luteinizing hormone (lutropin) may be administered to the subject torestore normal gonadal function. It is widely known in the art thatglycoprotein hormones such as FSH and LH are integral in femalereproductive physiology, and these glycoprotein hormones may beadministered to a subject to overcome a number of reproductive disordersand thereby assist reproduction.

[0075] Genetic therapy is another approach for treating hormonedisorders with the modified glycoprotein hormones of the presentinvention. In this approach, a gene encoding the modified glycoproteinhormone can be introduced into a cell, such as a germ line cell or asomatic cell, so that the gene is expressed in the cell and subsequentgenerations of those cells are capable of expressing the introducedgene. For example, any particular gonadotropin hormone can be insertedinto an ovarian cell, or its precursor, to enhance ovulation.Alternatively, introducing thyroid cells carrying a gene encoding asuperagonist of the thyroid stimulating hormone into an individual withthyroid carcinoma can obviate the need for continual administration ofTSH for stimulating radioiodine uptake in the thyroid carcinoma.Suitable vectors to deliver the coding sequence are well known in theart. For example, the vector could be viral, such as adenoviral,adenoassociated virus, retrovirus, or non-viral, such as cationicliposomes.

[0076] The modified glycoprotein hormones as provided by the presentinvention can also be used for targeting delivery of therapeutic agentsto thyroid tissues or gonadal tissue, or in the treatment of certainneoplasms.

[0077] In yet another aspect, the invention provides a method ofconstructing superactive nonchimeric analogs of human hormonescomprising comparing the amino acid sequence of a more active homologfrom another species to the human hormone, substituting amino acids inthe human hormone with the corresponding amino acids from the otherspecies, determining the activity of the substituted human hormone, andselecting superactive analogs from the substituted human hormones.Superactive analogs of human hormones includes any analog whose activityis increased over the corresponding activity of the wild-type hormone.For example, the modification of the human thyroid stimulating hormoneat position 11 in the α-subunit from threonine to lysine (T11K) resultsin a relative increase in the cAMP production in JP09 cells cultured invitro. (See Table II as set forth in the Example contained herein). Thismodification of the human thyroid stimulating hormone therefore resultsin a superactive analog of the wild-type human thyroid stimulatinghormone. The specific amino acid or amino acids to substitute to createthe modification can be determined, as discussed above, by: determiningthe activity of the homolog from another species and comparing thatactivity to the human hormone; then comparing the aligned sequences todetermine the amino acid sequence differences; then substituting theappropriate amino acid in the hormone from another species for the aminoacid at the corresponding position in the human hormone; thendetermining the activity of the modified human hormone by one of theabove-mentioned techniques; and then comparing the activity of themodified human hormones to the wild-type human hormone, therebyselecting the superactive analogs from the substituted human hormones.

[0078] All combinations of amino acid substitutions may be utilized toobtain a glycoprotein superagonist. For example, neutral amino acids canbe substituted for basic or acidic amino acids. Alternatively, basicamino acids can be substituted for acidic or neutral amino acids, oracidic amino acids may be substituted for neutral or basic amino acids.One skilled in the art will recognize, as discussed above, thatsubstitution of one amino acid for another can be at either the nucleicacid level in the nucleotide sequence that encodes the glycoproteinhormone or part of the glycoprotein hormone, or at the polypeptidelevel. Any human hormone can be modified by this method and itssuperactive analogs selected. In particular, the human hormone can be aglycoprotein hormone.

EXAMPLES

[0079] The sequence between Cys10 and Pro21 of the human α-subunit wasselected as the primary target for mutagenesis (FIG. 1). hCG-basedhomology modeling suggested that this region of the α-subunit is distantfrom the β-subunit in all glycoprotein hormones, contains severalsurface-exposed residues and includes a single turn of a 3₁₀-helixbetween Pro16 and Ser19¹. The human α-subunit differs from bovine inposition 11, 13, 16, 17 and 20 (FIG. 1a) and four of these changes arenonconservative (Thr11→Lys, Gln13→Lys, Pro16→Lys and Gln20→Lys). We usedPCR amplification to determine the sequence of the 11-20 region in theα-subunit of several primates including higher apes (commonchimpanzee—Pan troglodytes, orangutan—Pongo pygmaeus), lesser apes(gibbon—Hylobates sp.), Old World monkey (baboon—Papio anubis) andcompare them with previously known mammalian sequences including. rhesusmacaque (Macaca mulatta; Old World monkey), common marmoset (Callithrixjacchus; New World monkey) and human (FIG. 1a). Simultaneous comparisonof the sequences between different species suggested that basic residuesin this region were replaced relatively late in primate evolution. TheRhesus monkey α-subunit gene codes for Lys residues at positions 11, 16and 20 and an Arg residue at position 13^(2,) the baboon sequence codesfor Gln at position 16, whereas gibbon sequence contains only one weaklybasic imidazolium group of His at position 13 (FIG. 1a). Apparently acluster of positively charged amino acids in this region was maintainedand modified during vertebrate evolution, but is not present in thehigher apes and human sequence. The gradual elimination of positivelycharged residues in the 11-20 region of α-subunit coincide with theevolutionary divergence of the hominoids (human and apes) from the OldWorld monkeys. Our hypothesis that this region may modulate binding tothe receptor was further supported by: 1) the highest reactivity ofTyr21 in bTSH toward iodination³, 2) mapping of antigenic determinantsin hCG⁴, 3) the role of amino groups of Lys in the ovine and humanα-subunit for effective hormone-receptor interaction as studied byacylation⁵, labeling with acetic anhydride⁶ and pegylation of individualsubunits.

[0080] Consequently, positively charged Lys residues were inserted intothe Cys10-Pro21 region of the human α-subunit (FIG. 1b). Two otherregions were also mutagenized (Table I). A single nonconservativeLeu69→Arg mutation in the TSHβ-subunit was made based on a similarsequence comparison.

[0081] Effects of Mutations

[0082] Cotransfection of wild-type (WT) or mutant human α and hTSHβ⁷ orhCGβ cDNAs in various combination into CHO-K1 cells resulted in theexpression of 14 hTSH and 11 hCG heterodimers (Table I). In contrast tomany other mutagenesis studies^(8,9) the expression of mutants wasgenerally comparable to the WT. The following hTSH α-mutants wereexpressed at levels higher than WT-hTSH: T11K, Q13K, P16K, Q20K, Q50Pand Q13K+P16K+Q20K. Thus, this set of evolutionary justified mutationsdid not impair, in a major way, synthesis of the hTSH or hCG molecule,but may facilitate in certain cases hormone production.

[0083] Various bioassays were used to compare the relative potency andefficacy of hTSH and hCG mutants. The ability of WT and mutant hTSH tostimulate cAMP production was tested in CHO-JP09 cells with stablytransfected human TSH receptor. This assay revealed the following orderof potencies in single α-subunit mutants: P16K (6-fold lower EC₅₀ thanWT)≧Q20K>Q13K>T11K>WT-hTSH≈Q50P≈R67K (Table II). Receptor bindingactivity of WT and mutants hTSH was assessed in a competitive bindingassay to porcine thyroid membranes. Consistent with the cAMPstimulation, the following order of potencies was observed: P16K (5-foldgreater affinity than WT)>Q20K≧Q13K>T11K>WT-hTSH≈Q50P≈R67K (Table II).Thus, the increase in potency of single mutants observed in JP09 cellswas directly correlated with the increase of affinity to the TSHreceptor. Most notably, each mutation to a Lys residue in the 11-20region caused a substantial increase in activity, but changes outsidethis critical region had no (R67K, Q50P) effect on receptor bindingaffinity and bioactivity (Table II). Alanine mutagenesis of amino acids13, 16 and 20 in hTSH did not significantly alter hormone activity,indicating that only selective reconstitution of basic amino acidspresent in homologous hormones of other species resulted in thefunctional changes. Moreover, the exchange of αSer43 to Arg and thereplacements of αHis90 and αLys91 showed that these residues were lessimportant for hTSH than for hCG bioactivity, emphasizing hormone- andsite-specific roles of basic residues⁹.

[0084] Superagonists with Combined Mutations

[0085] To further study the effect of Lys residues which wereindividually responsible for highest increases in potency, mutantscontaining multiple replacements were produced. The most active mutantsare presented in FIGS. 2 and 3. The double Pro16→Lys+Gln20→Lys and thetriple Pro16→Lys+Gln20→Lys+Gln13-Lys mutants showed, respectively, 12and 24-fold higher activity than WT-hTSH, with a further increase inpotency up to 35-fold after Leu69-Arg replacement in the TSHβ-subunit(FIG. 2a). Additional optimization included substitution Glu14→Lys (Lysin this position present in the tuna sequence) resulted in furtherincrease in bioactivity up to 95-fold; these most potent multiplemutants elevated efficacy (maximal response) at least 1.5-fold (FIG.2b). These increases were verified by testing the ability of hTSHmutants to bind to porcine as well as human TSH receptor (Table II, FIG.2c and FIG. 2d), to induce growth in FRTL-5 cells (FIG. 2e), as well asT₃ production in cultured human thyroid follicles. In particular,Pro16→Lys+Gln20→Lys+Gln13→Lys/WT-hTSHβ andPro16→Lys+Gln20→Lys+Gln13→Lys/Leu69→Arg mutants required, respectively,18- and 27-fold lower concentration to attain half-maximal stimulationof ³H-thymidine incorporation in FRTL-5 cells than the WT-hTSH (FIG.2e). The synergistic effect of multiple mutations on TSH bioactivity wasnot limited to a local cooperation of Lys residues in the 13-20 regionof the α-subunit with receptor, but also involved the contribution ofArg69 in the opposite loop of β-subunit (Table II). TABLE I Relativeexpression of wild-type (WT) and mutant hormones in CHO-K1 cells hTSHhCG WT 100 ± 7 100 ± 4 T11K 267 ± 22  82 ± 2 Q13K 188 ± 9 106 ± 7 P16K206 ± 25  72 ± 6 Q20K 149 ± 18 117 ± 8 P16K + Q20K  86 ± 9  62 ± 6Q13K + P16K + Q20K 134 ± 6  76 ± 12 Q13K + P16K + Q20K + E14K  76 ± 12 52 ± 8 P16K + F17T  23 ± 10  93 ± 4 Q50P 174 ± 15  83 ± 3 R67K 171 ± 14 88 ± 6 β3-L69R  74 ± 5 n.a. Q13K + P16K + Q20K + β − L69R  86 ± 6 n.a.Q13K + P16K + Q20K + E14K + β− L69R  25 ± 6 n.a.

[0086] These findings were further confirmed in the animal model. Asingle injection of Pro16→Lys, Gln20→Lys and Gln13→Lys hTSH mutants inmice increased serum T₄ significantly higher than the WT-hTSH. Moreover,Pro16→Lys+Gln20→Lys+Gln13→Lys/WT-hTSHβ andPro16→Lys+Gln20→Lys+Gln13→Lys/Leu69→Arg mutants also generated higher T₄levels as compared to WT-hTSH (FIG. 2f). hTSH serum levels 6 h afteri.p. injection in mice were similar and the hTSH analogs did not showcompared to the WT great differences in the metabolic clearance rate.TABLE II The effects of site-specific mutagenesis of human glycoproteinhormones hTSH hTCG cAMP stimulation in JP09 cells Inhibition of¹²⁵I-bTSH Progesterone synthesis in MA10 EC₅₀ (ng/ml) Relative potency(WT = 1) binding (EC₂₅, ng/ml) cells (EC₅₀, ng/ml; Max, %) WT 6.70 ±0.69 1.0 81.3 ± 13.8 6.90 ± 1.04 100 ± 11 T11K 4.47 ± 0.79 1.5 68.3 ±4.4  2.79 ± 0.25 156 ± 23 Q13K 1.89 ± 0.41 3.5 22.5 ± 2.6  2.46 ± 0.28115 ± 24 P16K 1.05 ± 0.26 6.4 18.3 ± 3.6  2.05 ± 0.17 161 ± 31 Q20K 1.16± 0.22 5.8 21.3 ± 3.8  2.98 ± 0.27 134 ± 10 P16K + Q20K 0.57 ± 0.10 11.86.4 ± 2.4 1.70 ± 0.13 212 ± 34 Q13K + P16K + Q20K 0.28 ± 0.07 23.9 2.3 ±0.3 1.58 ± 0.09 216 ± 36 Q13K + P16K + Q20K + E14K 0.17 ± 0.04 39.4 2.1± 0.4 1.65 ± 0.06 205 ± 41 P16K + F17T 3.52 ± 0.50 1.9 n.d. n.d. n.d.Q50P 5.54 ± 0.70 1.2 77.5 ± 12.4 3.90 ± 0.85 137 ± 27 R67K 7.36 ± 0.330.9 62.5 ± 15.5 4.60 ± 0.63 145 ± 12 TSHβ-L69R 2.75 ± 0.49 2.4 n.d. n.a.n.a. TSHβ-L69R + +Q13K + P16K + Q20K 0.19 ± 0.06 35.3 1.8 ± 0.3 n.a.n.a. TSHβ-L69R + +Q13K + P16K + Q20K 0.07 ± 0.02 95.7 1.3 ± 0.4 n.a.n.a. bTSH 0.71 ± 0.14 9.4 7.9 ± 2.5 n.a. n.a.

[0087] A sequence comparison of the hCG and hTSH β-subunits showed aregion (residues 58-69 in TSHβ) which contains a cluster of basicresidues in hCG, but not in hTSH. We used site-directed mutagenesis tointroduce single and multiple basic residues into hTSH, based on theirlocation in hCG, generating the additional hTSH β-subunit mutants: I58R,E63R, I58R+E63R, 158R+E63R+L69R. The mutant hTSH β-subunits werecoexpressed with the human α-subunit and the intrinsic activity of therecombinant hTSH analogs studied at the rat THS receptor (FRTL-5 cells)and human TSH receptor (CHO-hTSHr cells). In both systems, singlesubstitutions (I58R, E63R) increased potency of hTSH 2-fold to 4-fold,and led to a slight increase of efficacy (FIG. 2g). The combination ofthe two substitutions (I58R+E63R) resulted in the potency which was15-fold higher than that of wild type hTSH and an 1.5-fold increase ofefficacy (FIG. 2g). Potency and efficacy of the combination mutantI58R+E63R+L69R, in which three basic residues were introduced, waselevated 50-fold and 1.7-fold, respectively (FIG. 2g). These increasesof intrinsic activity were accompanied by concomitant increases inreceptor binding affinity, judged by a receptor-binding assay usingCHO-JP09 cells. (FIG. 2h). Similarly, when mice were injected with theI58R+E63R+L69R mutant, their T₄ stimulation was significantly higherthan in either mock or control treated mice. (FIG. 2i).

[0088] The bioactivity of hCG mutants was tested using progesteronestimulation in MA-10 cells and cAMP stimulation in COS-7 cellstransfected with human LH/hCG receptor. hCG Lys mutants showed bothhigher potency (lower EC₅₀ values) as well as higher efficacy (V_(max))than the WT-hCG (Table II, FIG. 3a). The effect of single and multiplemutations was relatively analogous to that observed for the hTSHmutants. The αPro16→Lys/WT-hCGβ mutant was 4-fold more active thanWT-hCG in the stimulation of progesterone production and receptorbinding activity in MA-10 cells, with further increases in both potencyand efficacy for the Pro16→Lys+Gln20→Lys and Pro16→Lys+Gln20→LyshCG+Gln13→Lys mutants (FIGS. 3a and 3 b). Similar increases of intrinsicactivity were also found when studied at the human LH/hCG receptor(COS-7-hLH/hCG-R cells) (FIGS. 3c and 3 d).

[0089] Our data suggest that only a few amino acid replacements aresufficient to increase glycoprotein hormone bioactivity, even to a levelhigher than that of the model hormone (such as bTSH). Interestingly,only a few of the 40 differing residues between bovine and human TSHappear responsible for the higher biological activity of bTSH. Themajority of the other replacements are conservative, and as illustratedby the R67K mutation in the α-subunit, seem to have no functionalsignificance. In contrast, we show that surface-located Lys residuesclustered in the L1 loop and β1-strand of the α-subunit are crucial forthe high bioactivity of bTSH. Accordingly, recombinant hTSH with onlytwo mutated amino acids (P16K+Q20K) attains an intrinsic activitycomparable to bTSH (Table II). Moreover, triple, quadruplicate andquintuple hTSH mutants show even higher potency than bTSH. These datasuggest that the difference in activity between bTSH and hTSH is aresult of several amino acid changes, including replacements increasingactivity, but also others which may reduce biopotency of bTSH at thehTSH receptor.

[0090] Although, we cannot exclude a possibility that several receptorspecies would be made from a single transfected cDNA (by alternativesplicing from cryptic sites or by posttranslational modifications), thefact that similar differences in activity were observed in differentcell systems argues strongly against the importance of differentreceptor species in the increase in potency, efficacy and affinity ofthese analogs. Furthermore, there is compelling evidence that naturallyoccurring hormone isoforms with various carbohydrate residues exerttheir effect at the post-receptor level with no or minimal effect onreceptor binding affinity¹⁰. Since the wild type hormones and theiranalogs were characterized in multiple experimental systems, it ishighly probable that phenomenon of increased bioactivity described hereis a rule rather than exception related to particular cell-dependentvariant of the receptor.

[0091] Perspectives of Rational Design of Glycoprotein Hormone Analogs

[0092] Previous site-directed mutagenesis studies of glycoproteinhormones focused primarily on the highly conserved regions and residues,using such strategies as alanine scanning mutagenesis¹¹ or multiplereplacement approaches⁹. Several important studies were based on thecreation of chimeric subunits using cassette mutagenesis and/orrestriction fragment exchange^(12,13,14). Our strategy based onreplacement of nonconserved residues to those present in other specieshas been successful and permitted the generation of other glycoproteinhormone analogs, including hFSH mutants with increased bioactivity. Theparallel improvement of bioactivity of hTSH, hCG and hFSH byintroduction of basic residues in the 11-20 region of human α-subunitmay be related to the fact that this region is distant from theβ-subunit in the crystal structure based model of hCG and in ourhomology model of hTSH. The virtual identity of this area in both modelsas well as the observation that the antibodies binding to 11-26 regionare not greatly influenced by subunit combination¹⁵ suggest that thisdomain may function similarly in all the glycoprotein hormones. Once theα-subunit was successfully engineered to create more potent agonists ofhTSH, hCG or hFSH, the same paradigm was used to modify their respectiveβ-subunits to generate the ultimate superagonists of each glycoproteinhormone. For example, an additional replacement of a nonpolar Leu69 toArg in the TSHβ-subunit resulted in further increase of hTSHbioactivity. In addition, the plasma half-life of our analogs can bemodified regarding to specific therapeutic needs.

[0093] Further design and refinement of glycoprotein hormone analogswill include detailed three-dimensional structure of thehormone-receptor complexes. Although the exact structure of glycoproteinhormone receptors has not been solved, several models ofhormone-receptor interaction have been proposed^(15,16,17,18). Inaccordance with the recent model of Jiang et al.¹⁷ the L1 loop ofα-subunit may participate in the interaction with the transmembraneportion of the receptor. The cluster of positively charged residues inthis loop may enhance such an interaction and facilitate furtherrearrangements in the receptor leading to the activation of G proteinsand signal transduction.

[0094] Methods and Materials.

[0095] Restriction enzymes, DNA markers and other molecular biologicalreagents were purchased from either Gibco BRL (Gaithersburg, Md.) orfrom Boehringer-Mannheim (Indianapolis, Ind.). Cell culture media, fetalbovine serum and LipofectAMINE were purchased from Gibco BRL(Gaithersburg, Md.). VentR DNA Polymerase was purchased from New EnglandBiolabs (Beverly, Mass.). The full length human α cDNA (840 bp)subcloned into BamHI/XhoI sites of the pcDNA I/Neo vector (InvitrogenCorp., San Diego, Calif.) and hCG-β gene were obtained from Dr. T. H. Ji(University of Wyoming, Laramie, Wash.). The hTSH-β minigene without thefirst intron, with the non-translated 1st exon and authentic translationinitiation site was constructed in our laboratory. rhTSH-G standard wasfrom Genzyme Corp. (Framingham, Mass.). The CHO cells with stablyexpressed hTSH receptor (CHO-hTSHR clone JP09 and clone JP26) wereprovided by Dr. G. Vassart (University of Brussels, Brussels, Belgium).The human LH receptor cDNA was obtained from Dr. T. Minegishi (GunmaUniversity, Gunma, Japan). FRTL-5 cells were kindly supplied by Dr. L.D. Kohn (NIDDK, NIH, Bethesda, Md.). MA-10 cells were generouslysupplied by Dr. M. Ascoli (University of Iowa, Iowa City, Iowa). ¹²⁵IcAMP and ¹²⁵I-hTSH were from Hazleton Biologicals (Vienna, Va.). Bloodsamples of various primates were obtained from Yerkes Regional PrimateResearch Center (Emory University, Atlanta, Ga.) and Animal Resources(University of Oklahoma, Oklahoma City, Okla.).

[0096] Determination of Primate α-Subunit Sequences

[0097] The QIAamp^(R) Blood Kit (Qiagen Inc., Chatsworth, Calif.) wasused for extraction of genomic DNA from whole blood samples ofchimpanzee (Pan troglodytes), orangutan (Pongo pygmaeus), gibbon(Hylobates sp.) and baboon (Papio anubis). Genomic DNA was used in thePCR; the synthetic oligonucleotide primers used were

[0098] 5′-CCTGATAGATTGCCCAGAATGC-3′ (sense) (SEQ ID NO:1) and

[0099] 5′-GTGATAATAACAAGTACTGCAGTG-3′ (antisense) (SEQ ID NO:2)

[0100] and were synthesized according to the nucleotide sequence of thegene encoding common α-subunit of human glycoprotein hormones¹⁹. PCR wasperformed using 800-1000 ng of genomic DNA template and 10 picomoles ofeach primer in 100 μl reaction volume that also contained 10 mMTris-HCl, (pH 9.0 at 25° C.), 50 mM KCl, 2.5 mM MgCl₂, 200 μM dNTPs and2 U of Taq DNA Polymerase (Promega Corp. Madison, Wis.). The reactionmix was covered with mineral oil, and each sample was initially heatedto 95° C. for 10 min. The PCR program consisted of 32 cycles ofdenaturation at 95° C. for 1 min 30 sec, annealing at 55° C. for 1 min30 sec and extension at 72° C. for 1 min, followed by a final extensionperiod at 72° C. for 7 min. The reactions were then directlyelectrophoresed on a 1% agarose gel in the presence of ethidium bromide.The amplified PCR product (˜700 bp), spanning the nucleotide sequence ofexon 3, intron 3 and exon 4, was purified using QIAquick PCRPurification Kit (QIAGEN Inc., Chatsworth, Calif.) and subcloned intopCR™II using Original TA Cloning Kit (Invitrogen Corp., San Diego,Calif.). The sequence of the fragment was obtained after subcloning ordirect dideoxy sequencing using a Sequenase kit (U.S. Biochemical Corp.,Cleveland, Ohio).

[0101] Homology Modeling.

[0102] Modeling relies on the strong sequence homology between hCG andhTSH. The sequences were aligned to bring the cysteine-knot residuesinto correspondence and the percentage of identical as well as highlyconservative replacements were calculated as described¹. There was 58%sequence identity between hCG and hTSH molecules; 31% of the twoβ-subunit sequences were identical and additional 17% included highlyconservative changes in β-subunit. A molecular model of hTSH was builton a template of hCG model derived from crystallographic coordinatesobtained from the Brookhaven Data Bank²⁰. All coordinate manipulationsand energy calculations were done using CHARMm release 21.2 for theConvex and further modified using the molecular graphic package QUANTA(Version 3.3, Molecular Simulations Inc., University of York, York,United Kingdom).

[0103] Site-Directed Mutagenesis.

[0104] Mutagenesis of the human α-cDNA and the hTSHβ minigene wasaccomplished by the PCR-based megaprimer method²¹. Amplification wasoptimized using Vent^(R) DNA Polymerase (New England Biolabs, Beverly,Mass.). After digestion with BamHI and XhoI PCR product was ligated intopcDNA I/Neo (Invitrogen Corp., San Diego, Calif.) with the BamI/XhoIfragment excised. MC1061/p3 E. coli cells were transformed usingUltracomp E. coli Transformation Kit (Invitrogen Corp.). The QIAprep 8Plasmid Kit (QIAGEN Inc., Chatsworth, Calif.) was used for multipleplasmid DNA preparations. QIAGEN Mega and Maxi Purification Protocolswere used to purify larger quantities of plasmid DNA. Multiple mutantswere created with the same method using plasmids containing α-cDNA witha single mutation as a template for further mutagenesis. Mutations wereconfirmed by double stranded sequencing using Sanger's dideoxynucleotidechain termination procedure.

[0105] Expression of Recombinant Hormones.

[0106] CHO-K1 Cells (ATCC, Rockville, Md.) were maintained in Ham's F-12medium with glutamine and 10% FBS, penicillin (50 units/mil) andstreptomycin (50 μg/ml). Plates of cells (100 mm culture dishes) werecotransfected with wild type or mutant α-cDNA in the pcDNAI/NEO andhTSHβ minigene inserted into the p(LB)CMV vector⁷, or pcDNAI/Neocontaining hCGβ-cDNA⁸ using a LipofectAMINE (Gibco BRL, Gaithersburg,Md.). After 24 h, the transfected cells were transferred to CHO-serumfree medium (CHO-SFM-II, Gibco BRL,). The culture media includingcontrol medium from mock transfections using the expression plasmidswithout gene inserts were harvested 72 h after transfection,concentrated and centrifuged; the aliquots were stored at −20° C. andthawed only once before each assay. WT and mutant hTSH were measured andverified using four different immunoassays as described⁹. Concentrationsof WT and mutant hCG were measured using chemiluminescence assay (hCGKit, Nichols Institute, San Juan Capistrano, Calif.) andimmunoradiometric assay (hCG IRMA, ICN, Costa Mesa, Calif.).

[0107] cAMP Stimulation in JP09 Cells Expressing Human TSH Receptor.

[0108] CHO cells stably transfected with hTSH receptor cDNA (JP09) weregrown and incubated with serial dilutions of WT and mutant hTSH asdescribed⁹. cAMP released into the medium was measured byradioimmunoassay²². The equivalent amounts of total media protein wereused as the mock control and the hTSH containing samples fromtransfected cells.

[0109] cAMP Stimulation in COS-7 Cells Expressing Human LH Receptor.

[0110] COS-7 cells transiently transfected with hLH receptor cDNA weregrown and incubated with serial dilutions of WT and mutant hCGessentially as described²³. cAMP released into the medium was measuredby radioimmunoassay²². The equivalent amounts of total media proteinwere used as the mock control and the hCG containing samples fromtransfected cells.

[0111] Progesterone Production Stimulation in MA-10 Cells.

[0112] Transformed murine Leydig cells (MA-10) grown in 96-well cultureplates were incubated with WT and mutant hCG for 6 hours in the assaymedium as described²⁴. The amount of progesterone released into themedium was determined by radioimmunoassay (CT Progesterone Kit, ICNBiomedicals, Inc., Costa Mesa, Calif.).

[0113] Receptor Binding Assays.

[0114] The receptor-binding activities of hTSH analogs were assayed bytheir ability to displace ¹²⁵I-bTSH from a solubilized porcine thyroidmembranes²²⁴. The binding activities of selected analogs to human TSHreceptor was tested using JP09 cells. The binding activities of hCGanalogs to MA-cells and to COS-7 cells transiently transfected withhuman LH receptor were determined using ¹²⁵I-hCG and assay medium asdescribed previously²⁴.

[0115] Thymidine Uptake Stimulation in FRTL-5 Cells.

[0116] Growth of the rat thyroid cells (FRTL-5) was monitored aspreviously described²².

[0117] Stimulation of T₄ Secretion in Mice.

[0118] The in vivo bioactivity of the WT and mutant TSH was determinedusing a modified McKenzie bioassay^(22,25). WT and mutant TSH wereinjected i.p. into male albino Swiss Cr1:CF-1 mice with previouslysuppressed endogenous TSH by administration of 3 μg/ml T₃ in drinkingwater for 6 days. Blood samples were collected 6 h later from orbitalsinus and the serum T₄ and TSH levels were measured by respectivechemiluminescence assays (Nichols Institute).

[0119] Throughout this application various publications are referenced.Certain publications are referenced by numbers within parentheses. Fullcitations for the number-referenced publications are listed below. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

[0120] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

REFERENCES

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[0127] 7. Joshi, L. et al. Recombinant thyrotropin containing aβ-subunit chimera with the human chorionic goandotropin-β carboxyterminus is biologically active, with a prolonged plasma half-life: roleof carbohydrate in bioactivity and metabolic clearance. Endocrinology136:3839-3848 (1995).

[0128] 8. Ji, I., Zeng H. & Ji; T. H. J. Receptor activation of andsignal generation by the lutropin/choriogonadotropin receptor. Biol.Chem. 268:22971-22974 (1993).

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[0134] 14. Dias, J. A., Zhang, Y. and Liu, X. Receptor binding andfunctional properties of chimeric human follitropin prepared by anexchange between a small hydrophilic intercysteine loop of humanfollitropin and human lutropin. J. Biol. Chem. 269:25289-25294 (1994).

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1 37 22 base pairs nucleic acid single linear DNA (genomic) 1 CCTGATAGATTGCCCAGAAT GC 22 24 base pairs nucleic acid single linear DNA (genomic)2 GTGATAATAA CAAGTACTGC AGTG 24 12 amino acids amino acid Not Relevantlinear peptide 3 Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro 1 5 1012 amino acids amino acid Not Relevant linear peptide 4 Cys Thr Leu GlnGlu Asn Pro Phe Phe Ser Gln Pro 1 5 10 12 amino acids amino acid NotRelevant linear peptide 5 Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser GlnPro 1 5 10 12 amino acids amino acid Not Relevant linear peptide 6 CysGln Leu His Glu Asn Pro Phe Phe Ser Gln Pro 1 5 10 12 amino acids aminoacid Not Relevant linear peptide 7 Cys Lys Pro Arg Glu Asn Gln Phe PheSer Lys Pro 1 5 10 12 amino acids amino acid Not Relevant linear peptide8 Cys Lys Pro Arg Glu Asn Lys Phe Phe Ser Lys Pro 1 5 10 12 amino acidsamino acid Not Relevant linear peptide 9 Cys Lys Leu Lys Glu Asn Lys TyrPhe Ser Arg Leu 1 5 10 12 amino acids amino acid Not Relevant linearpeptide 10 Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro 1 5 10 12amino acids amino acid Not Relevant linear peptide 11 Cys Lys Leu LysGlu Asn Lys Tyr Phe Ser Lys Pro 1 5 10 12 amino acids amino acid NotRelevant linear peptide 12 Cys Lys Leu Arg Glu Asn Lys Tyr Phe Phe LysLeu 1 5 10 12 amino acids amino acid Not Relevant linear peptide 13 CysLys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu 1 5 10 12 amino acids aminoacid Not Relevant linear peptide 14 Cys Lys Leu Lys Glu Asn Lys Tyr PheSer Lys Leu 1 5 10 12 amino acids amino acid Not Relevant linear peptide15 Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Leu 1 5 10 12 amino acidsamino acid Not Relevant linear peptide 16 Cys Lys Leu Lys Glu Asn LysTyr Phe Ser Lys Leu 1 5 10 12 amino acids amino acid Not Relevant linearpeptide 17 Cys Lys Leu Lys Gln Asn Lys Tyr Phe Ser Lys Leu 1 5 10 12amino acids amino acid Not Relevant linear peptide 18 Cys Lys Leu GlyGlu Asn Arg Phe Phe Ser Lys Pro 1 5 10 12 amino acids amino acid NotRelevant linear peptide 19 Cys Lys Leu Gly Glu Asn Arg Phe Phe Ser LysPro 1 5 10 12 amino acids amino acid Not Relevant linear peptide 20 CysLys Leu Gly Glu Asn Arg Phe Phe Ser Lys Pro 1 5 10 12 amino acids aminoacid Not Relevant linear peptide 21 Cys Arg Leu Lys Glu Asn Leu Arg PheSer Asn Met 1 5 10 12 amino acids amino acid Not Relevant linear peptide22 Cys Lys Leu Lys Glu Asn Lys Val Phe Ser Asn Pro 1 5 10 12 amino acidsamino acid Not Relevant linear peptide 23 Cys Thr Leu Lys Pro Asn ThrIle Phe Pro Asn Pro 1 5 10 12 amino acids amino acid Not Relevant linearpeptide 24 Cys Lys Leu Lys Glu Asn Asn Ile Phe Ser Lys Pro 1 5 10 12amino acids amino acid Not Relevant linear peptide 25 Cys Thr Leu LysLys Asn Asn Val Phe Ser Arg Asp 1 5 10 12 amino acids amino acid NotRelevant linear peptide 26 Cys Thr Leu Arg Lys Asn Thr Val Phe Ser ArgAsp 1 5 10 12 amino acids amino acid Not Relevant linear peptide 27 CysThr Leu Arg Lys Asn Ser Val Phe Ser Arg Asp 1 5 10 12 amino acids aminoacid Not Relevant linear peptide 28 Cys Lys Leu Lys Glu Asn Asn Ile PheSer Lys Pro 1 5 10 12 amino acids amino acid Not Relevant linear peptide29 Cys Thr Leu Lys Glu Asn Asn Ile Phe Ser Lys Pro 1 5 10 12 amino acidsamino acid Not Relevant linear peptide 30 Cys Arg Leu Lys Asp Asn LysPhe Phe Ser Lys Pro 1 5 10 12 amino acids amino acid Not Relevant linearpeptide 31 Cys Arg Leu Gln Glu Asn Lys Ile Phe Ser Lys Pro 1 5 10 12amino acids amino acid Not Relevant linear peptide 32 Cys Thr Leu LysGlu Asn Pro Phe Phe Ser Gln Pro 1 5 10 12 amino acids amino acid NotRelevant linear peptide 33 Cys Thr Leu Gln Glu Asn Lys Phe Phe Ser GlnPro 1 5 10 12 amino acids amino acid Not Relevant linear peptide 34 CysThr Leu Gln Glu Asn Pro Phe Phe Ser Lys Pro 1 5 10 12 amino acids aminoacid Not Relevant linear peptide 35 Cys Thr Leu Gln Glu Asn Lys Phe PheSer Lys Pro 1 5 10 12 amino acids amino acid Not Relevant linear peptide36 Cys Thr Leu Lys Glu Asn Lys Phe Phe Ser Lys Pro 1 5 10 12 amino acidsamino acid Not Relevant linear peptide 37 Cys Thr Leu Lys Lys Asn LysPhe Phe Ser Lys Pro 1 5 10

What is claimed is:
 1. A modified human glycoprotein hormone comprisingat least three basic amino acids in the α-subunit at positions selectedfrom the group consisting of positions 11, 13, 14, 16, 17, and
 20. 2.The modified human glycoprotein hormone of claim 1, further comprising afourth basic amino acid at a position selected from the group consistingof positions 11, 13, 14, 16, 17, and
 20. 3. The modified humanglycoprotein hormone of claim 2, wherein basic amino acids are atpositions 11, 13, 16, and
 20. 4. The modified human glycoprotein ofclaim 2, wherein basic amino acids are at positions 11, 13, 17, and 20.5. The modified human glycoprotein hormone of claim 2, wherein basicamino acids are at positions 13, 14, 16, and
 20. 6. The modified humanglycoprotein hormone of claim 2, wherein basic amino acids are atpositions 13, 14, 17, and
 20. 7. The modified human glycoprotein hormoneof claim 2, further comprising a fifth basic amino acid at a positionselected from the group consisting of positions 11, 13, 14, 16, 17, and20.
 8. The modified human glycoprotein hormone of claim 7, wherein basicamino acids are at positions 13, 14, 16, 17, and
 20. 9. The modifiedhuman glycoprotein hormone of claim 7, wherein basic amino acids are atpositions 11, 13, 14, 16 and
 20. 10. The modified human glycoproteinhormone of claim 1, wherein basic amino acids are at positions 11, 13,14, 16, 17, and
 20. 11. The modified human glycoprotein hormone of claim1, wherein basic amino acids are at positions 13, 16, and
 20. 12. Themodified human glycoprotein hormone of claim 1, wherein the hormone isthyroid stimulating hormone.
 13. The modified human thyroid stimulatinghormone of claim 12, wherein the modified hormone further comprises abasic amino acid in at least one position selected from the groupconsisting of positions 58, 63, and 69 of the b-subunit.
 14. Themodified human thyroid stimulating hormone of claim 13, wherein themodified hormone comprises basic amino acids at positions 58, 63, and 69of the b-subunit.
 15. The modified human glycoprotein hormone of claim13, wherein a basic amino acid is at position
 58. 16. The modified humanglycoprotein hormone of claim 13, wherein a basic amino acid is atposition
 63. 17. The modified human glycoprotein hormone of claim 13,wherein a basic amino acid is at position
 69. 18. The modified humanglycoprotein hormone of claim 1, wherein the hormone isfollicle-stimulating hormone.
 19. The modified human glycoproteinhormone of claim 18, wherein the modified human glycoprotein hormonecomprises a basic amino acid in at least one position selected from thegroup consisting of positions in the β-subunit of glycoprotein hormones,corresponding to positions 58, 63, and 69 of the β-subunit of the humanthyroid stimulating hormone.
 20. The modified human glycoprotein hormoneof claim 1, wherein the hormone is luteinizing hormone.
 21. The modifiedhuman glycoprotein hormone of claim 20, wherein the modified humanglycoprotein hormone comprises a basic amino acid in at least oneposition selected from the group consisting of positions in theβ-subunit of glycoprotein hormones, corresponding to positions 58, 63,and 69 of the β-subunit of the human thyroid stimulating hormone. 22.The modified human glycoprotein hormone of claim 1, wherein the hormoneis chorionic gonadotropin.
 23. The modified human glycoprotein hormoneof any of claim 22, wherein the modified human glycoprotein hormonecomprises a basic amino acid in at least one position selected from thegroup consisting of positions in the β-subunit of glycoprotein hormones,corresponding to positions 58, 63, and 69 of the β-subunit of the humanthyroid stimulating hormone.
 24. The modified human glycoprotein hormoneof claim 1, wherein the basic amino acids are lysine.
 25. The modifiedhuman glycoprotein hormone of claim 1, wherein the basic amino acids areselected from the group consisting of lysine and arginine.
 26. A methodof assisting reproduction in a subject comprising administering anassisting amount of the modified glycoprotein hormone of claim
 1. 27. Anucleic acid encoding the modified human glycoprotein hormone ofclaim
 1. 28. A vector comprising the nucleic acid of claim 27, whereinthe vector is suitable for expressing the nucleic acid.
 29. A hostcomprising the vector of claim 28, wherein the host is suitable forexpressing the nucleic acid.
 30. The modified human glycoprotein hormoneof claim 1 further modified so that the modified human glycoproteinhormone has less than five amino acid substitutions in the α-subunit inpositions other than positions 11, 13, 14, 16, 17, and
 20. 31. Themodified glycoprotein hormone of claim 1, wherein the modified humanglycoprotein hormone has less than four amino acid substitutions in thea-subunit in positions other than positions 11, 13, 14, 16, 17, and 20.32. The modified glycoprotein hormone of claim 1, wherein the modifiedhuman glycoprotein hormone has less than three amino acid substitutionsin the a-subunit in positions other than positions 11, 13, 14, 16, 17,and
 20. 33. The modified glycoprotein hormone of claim 1, wherein themodified human glycoprotein hormone has less than two amino acidsubstitutions in the a-subunit in positions other than positions 11, 13,14, 16, 17, and
 20. 34. The modified glycoprotein hormone of claim 1,wherein the hormone human glycoprotein hormone has the complete aminoacid sequence homology with the human glycoprotein hormone in positionsother than positions 11, 13, 14, 16, 17, and 20 of the a-subunit.
 35. Amodified human glycoprotein hormone comprising a basic amino acid in thea-subunit in at least one position selected from the group consisting ofpositions 11, 13, 14, 16, 17 and
 20. 36. The modified human glycoproteinhormone of claim 35, wherein a basic amino acid is at position
 11. 37.The modified human glycoprotein hormone of claim 35, wherein a basicamino acid is at position
 11. 38. The modified human glycoproteinhormone of claim 35, wherein a basic amino acid is at position
 14. 39.The modified human glycoprotein hormone of claim 35, wherein a basicamino acid is at position
 16. 40. The modified human glycoproteinhormone of claim 35, wherein a basic amino acid is at position
 17. 41.The modified human glycoprotein hormone of claim 35, wherein a basicamino acid is at position
 20. 42. The modified human glycoproteinhormone of claim 35, wherein the basic amino acid is selected from thegroup consisting of lysine and arginine.
 43. The modified humanglycoprotein hormone of claim 35, comprising a basic amino acid in atleast two positions selected from the group consisting of positions 11,13, 14, 16, 17, and
 20. 44. The modified human glycoprotein hormone ofclaim 42, wherein the basic amino acids are at positions 16 and
 20. 45.The modified human glycoprotein hormone of claim 42, wherein the basicamino acids are at positions 16 and
 13. 46. The modified humanglycoprotein hormone of claim 42, wherein the basic amino acids are atpositions 20 and
 13. 47. The modified human glycoprotein hormone ofclaim 42, wherein the basic amino acid is selected from the groupconsisting of lysine and arginine.
 48. The modified glycoprotein hormoneof claim 35, wherein the modified hormone is thyroid stimulatinghormone.
 49. The modified human thyroid stimulating hormone of claim 48,wherein the modified hormone further comprises a basic amino acid in atleast one position selected from the group consisting of positions 58,63, and 69 of the b-subunit.
 50. The modified human thyroid stimulatinghormone of claim 49, wherein the modified hormone comprises basic aminoacids at positions 58, 63, and 69 of the b-subunit.
 51. The modifiedhuman glycoprotein of claim 49, wherein a basic amino acid is atposition
 58. 52. The modified human glycoprotein of claim 49, wherein abasic amino acid is at position
 63. 53. The modified human glycoproteinof claim 49, wherein a basic amino acid is at position
 69. 54. Themodified glycoprotein of claim 35, wherein the hormone isfollicle-stimulating hormone.
 55. The modified glycoprotein of claim 35,wherein the hormone is luteinizing hormone.
 56. The modifiedglycoprotein of claim 35, wherein the hormone is chorionic gonadotropin.57. A method of treating a condition associated with a glycoproteinhormone activity in a subject comprising administering an effectiveamount of a suitable modified glycoprotein hormone of claim 1 to thepatient.
 58. The method of claim 56, wherein the condition is ovulatorydisfunction.
 59. The method of claim 56, wherein the condition is aluteal phase defect.
 60. The method of claim 56, wherein the conditionis unexplained infertility.
 61. The method of claim 56, wherein thecondition is male factor infertility.
 62. The method of claim 56,wherein the condition is time-limited conception.
 63. The method ofclaim 56, wherein the condition is thyroid carcinoma.
 64. A method ofassisting reproduction in a subject comprising administering anassisting amount of the modified glycoprotein hormone of claim
 35. 65. Anucleic acid encoding the modified human glycoprotein of claim
 35. 66. Avector comprising the nucleic acid of claim 65, wherein the vector issuitable for expressing the nucleic acid.
 67. A host comprising thevector of claim 66, wherein the host is suitable for expressing thenucleic acid.
 68. The modified human glycoprotein hormone of claim 35further modified so that the modified human glycoprotein hormone havingless than five amino acid substitutions in the α-subunit in positionsother than positions 11, 13, 14, 16, 17, and
 20. 69. The modifiedglycoprotein hormone of claim 35, wherein the modified humanglycoprotein hormone has less than four amino acid substitutions in thea-subunit in positions other than positions 11, 13, 14, 16, 17, and 20.70. The modified glycoprotein hormone of claim 35, wherein the modifiedhuman glycoprotein hormone has less than three amino acid substitutionsin the a-subunit in positions other than positions 11, 13, 14, 16, 17,and
 20. 71. The modified glycoprotein hormone of claim 35, wherein themodified human glycoprotein hormone has less than two amino acidsubstitutions in the a-subunit in positions other than positions 11, 13,14, 16, 17, and
 20. 72. The modified glycoprotein hormone of claim 35,wherein the modified human glycoprotein hormone has the complete aminoacid sequence homology with the wild-type human glycoprotein hormone inpositions other than positions 11, 13, 14, 16, 17, and 20 of thea-subunit.
 73. The modified human glycoprotein hormone of claim 35,wherein the modified human glycoprotein hormone comprises a basic aminoacid in at least one position selected from the group consisting ofpositions in the b-subunit of glycoprotein hormones other than thethyroid stimulating hormone, corresponding to positions 58, 63, and 69of the b-subunit of the human thyroid stimulating hormone.
 74. Amodified human glycoprotein hormone having increased activity over thecorresponding wild-type glycoprotein hormone, wherein the modified humanglycoprotein hormone comprises a basic amino acid substituted at aposition corresponding to the homologous amino acid position in a moreactive non-human glycoprotein hormone homolog.
 75. A modifiednonchimeric glycoprotein hormone from a particular species havingincreased activity over the correspondig wild-type glycoprotein hormone,wherein the modified glycoprotein hormone comprises a basic amino acidsubstituted at a position corresponding to the homologous amino acidposition in a more active glycoprotein hormone homolog from anotherspecies.
 76. The modified human glycoprotein hormone of claim 74,wherein the non-human glycoprotein hormone homolog is a bovineglycoprotein hormone.
 77. The modified human glycoprotein hormone ofclaim 74, wherein the basic amino acid is lysine.
 78. The modified humanglycoprotein hormone of claim 74, wherein the activity of the modifiedhuman glycoprotein hormone is increased by at least 3 fold.
 79. A methodof constructing superactive nonchimeric analogs of human glycoproteinhormones comprising: a) comparing the amino acid sequence of a moreactive homolog from another species to the human glycoprotein hormone;b) substituting amino acids in the human glycoprotein hormone with thecorresponding amino acids from the homolog of the other species; c)determining the activity of the substituted human glycoprotein hormone;and d) selecting superactive analogs from the substituted humanglycoprotein hormones.
 80. A method of constructing antagonistnonchimeric analogs of human glycoprotein hormones, comprising: a)comparing the amino acid sequence of a less active homolog from anotherspecies to the human glycoprotein hormone; b) substituting amino acidsin the human glycoprotein hormone with the corresponding amino acidsfrom the other species; c) determining the activity of the substitutedhuman hormone; and d) selecting antagonist analogs from the substitutedhuman hormones.